JPS63124963A - Detection of adult t cell leukemia virus antibody - Google Patents

Abstract

PURPOSE:To improve the accuracy of detection by using the polypeptide coded by the blended gene conjugated with the gag gene of adult T cell leukemia virus (ATLV) or the part thereof and env gene of ATLV or the part thereof as an antigen. CONSTITUTION:This polypeptide used as the antigen is formed by bonding the polypeptide which is composed of amino acids from the 14th to the 139th at the N terminal side in the P-24 produced from the gag gene with the polypeptide which is composed of amino acids from the 197th to the 295th at the N terminal side in the env gene, and is respectively added with methionine-aspartic acid to the N terminal side and lysine to the C terminal side thereof. The anti- ATLV antibody in serum is detected by a sandwich method using such polypeptide. The anti-ATLV antibody is thereby detected with the high accuracy.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is directed to adult T-cell leukemia virus (abbreviated as ATLV, but also referred to as human T-cell leukemia virus (HTLV-1)). The present invention relates to a method for detecting anti-ATLV antibodies in a sample by an immunological method using as an antigen substance a polypeptide encoded by a fusion gene in which the ATLV gag gene or a part thereof is combined with the ATLV env gene or a part thereof.

Therefore, the present invention is useful in the field of clinical diagnosis.

BACKGROUND OF THE INVENTION Conventionally, antigen-antibody reactions have been used in a wide range of fields, particularly in the medical field, where they are widely applied to the diagnosis, treatment, and prevention of diseases.

They rely on the high specificity of antigen-antibody reactions, which guarantees diagnostic accuracy and therapeutic and prophylactic effectiveness.

Adult T-cell leukemia (hereinafter referred to as ATL) is ATLV (
HTLV-1) is thought to be the cause, and ATLV (
Serological diagnosis of HTLV-1) infection is important for diagnosis of ATL and prevention of infection. ΔTLV infection is carried out by detecting the presence or absence of antibodies against ATL-related antigen (hereinafter referred to as ATLA) in serum using an antigen-antibody reaction. A conventionally known method for detecting anti-ATLV antibodies is a method using P-24, a product of the ATLV gag gene, as an antigen substance (hereinafter referred to as P-24).
method), a method using a polypeptide which is a gene product of whole ATLV as an antigenic substance, and a method using a polypeptide which is a gene product of whole ATLV as an antigenic substance;
Methods using nv gene products are known.

There are some problems that the invention attempts to solve, and it is desired to develop a more excellent detection method.

Means for Solving the Problems As a result of research to find an excellent method for detecting anti-ATLV antibodies, the present inventor discovered that the gag gene of ATLV and en
Anti-ATLV when a polypeptide encoded by a fusion gene linked to the
The present invention was completed based on the discovery that antibody detection can be performed with extremely high accuracy.

The present invention will be explained in detail below.

The present invention combines the gag gene of ATLV or a part thereof and A.
A method for detecting anti-ATLV antibodies, which comprises detecting anti-ATLV antibodies in a sample by an immunological method using a polypeptide encoded by a fusion gene bound to the TLV env gene or a part thereof as an antigenic substance. Regarding.

Polypeptides used as antigenic substances in the present invention include:
Any gene encoded by a fusion gene in which the gag gene or part thereof and the env gene or part thereof can be used. Preferably, a protein having the following amino acid sequence produced by the method shown in Reference Example 1 (hereinafter referred to as gag-env protein) is used. gag-env protein is P-24ON terminal side 1
A polypeptide in which a polypeptide from the 4th amino acid (proline) to the 139th amino acid (glycine) is combined with a polypeptide from the 197th amino acid (threonine) to the 295th amino acid (proline) on the N-terminal side of env. It is a polypeptide in which methionine-aspartic acid is added to the N-terminal side and lysine (Lys) is added to the C-terminal side.

Samples to which the present invention is applied include body fluids such as blood and urine.

Immunological methods used in the present invention include the following, and can be carried out using methods described in various literatures.

0 enzyme immunoassay (hereinafter referred to as EIΔ) Enzyme immunoassay, Igaku Shoin 1 Eiji Ishikawa et al.

1978.

0 Radio Immunoassay (hereinafter referred to as RIA) Immune Serology, Ishiyaku Publishing, Maya Inai et al., 1981 0 Fluorescence Immunoassay (hereinafter referred to as FIA) Same as above 0 Reverse passive agglutination reaction Same as above 0 Phytohaematoa Same as above 0 Laser nephrometry Same as above 0 Immune hemolysis reaction (method using red blood cells or liposomes) Same as above 0 Latest test for particle agglutination, Idensha, Vol. 2 N (12,151~
157°1984, Gann 75.845.19
In 84RIA, EIA, and FIA, not only homogeneous analysis methods but also heterogeneous analysis methods such as the Sand-Deutsch method are possible.

As a specific example, an example in which EIA is performed by the Santoifuchi method is shown below.

The antigenic material is coated onto the wells of a microtiter plate, and the serum sample is mixed with a suitable buffer such as Buffer A.
C0,15M NaCj! , O, '4% gelatin,
l containing 1% bovine serum albumin and 0.1% NaN3
/15M sodium phosphate buffer (pH 7,2))
Pour the diluted solution into the well and incubate at 4-37°C for 1 hour to 1 hour.
Leave to stand for a week, then add an appropriate buffer, such as buffer B (0,0
l/15M Sodium Phosphate 111 fresh solution containing 5% Tween 20 and 0.15M NaCR (pH 7,2
Wash the wells thoroughly with )). A conjugate of a rabbit anti-human immunoglobulin antibody Fab fragment and horseradish peroxidase conjugated by the periodic acid method is diluted with an appropriate buffer, such as buffer B, and then placed in a well.

After standing at 4 to 45°C for 1 to 24 hours, the plate is washed with the same buffer. A suitable substrate solution is then prepared, such as ortho-phenylenediamine substrate solution (0.02%).

l/15M containing ortho-phenylenediamine and 35mM hydrogen peroxide! Sodium phosphate buffer (pH 7,
2))) is placed in a well and left standing at 15-37°C for 5-60 minutes. The reaction was stopped with 2NH2S○, and 1~
After standing for 10 minutes, absorbance at 4101 m and 600 nm (
0, D, .), and the value obtained by subtracting the latter value from the former value is defined as the antibody value.

The method shown here is just an example, and the antibody, substrate solution, coloring agent, etc. can be changed as appropriate.

The method of the present invention using gag-env protein provides excellent anti-AT
This is a method for detecting LA antibodies, but gag gene products and en
Excellent effects similar to the method of the present invention can be expected even when using a product in which the V gene product is bound to the same carrier (hereinafter referred to as cocktail antigen polypeptide). A cocktail antigen polypeptide can be produced by physically or chemically adsorbing the gag gene product and the env gene product onto a suitable carrier, such as a liposome, a synthetic resin, or a solid support made of glass.

Although the method of the present invention shows excellent effects in detecting anti-ATLV antibodies, it is also effective in detecting TSP (Tropical 5pastic
araplesia, tropical spastic paraplegia), HA
M (HTLV-1 related myelopathy), MS (Mul
It can be applied to multiple sclerosis (tiple sclerosis, multiple sclerosis) and various cancers other than ATL (eg, liver cancer, uterus 1).

Example 1 EIA using gag-env protein (gag-env method) or P-24, which is a fragment of the gag gene product, as an antigen substance (P-24 method) and Eisai method using natural ATL (Atest ATL) Anti-ATLV antibodies were detected in the serum of ATL patients, HTLV-1 carriers, and normal subjects.

(1) g a g -e n v protein and P-2
EIA (Sand-Deutsch method) using 4 ATL patients or HTLV-1 carriers 5 as serum samples
Sera from 7 people and 30 normal people were used. gag-en
As the v protein, a polypeptide obtained by the method of Reference Example 1 was used, and as P-24, a gag polypeptide obtained according to the method described in JP-A-60-61534 was used.

One gram of gag-env protein and 3 Jtg of P-24 were each coated onto the wells of a microtiter plate (Nunc).

Each serum was diluted with buffer A (0.15M NaCl 1°0.1%).
Gelatin, 1% bovine serum albumin and 0.1% NaN
The mixture was diluted 20 times with 1/15M sodium phosphate buffer containing 5.5 (pH 7.2). This diluted solution 1001IJl was added to the above well, and after standing at room temperature for 2 hours, t! i buffer solution B
CO105% Tween 20 (manufactured by Wako Pure Chemical Industries, Ltd.)
and 0.15M Na (1/15M sodium phosphate buffer containing J, pH 7,2) 150jt! Washed 3 times with l.

Fab fragment of rabbit anti-vito immunoglobulin antibody (enzyme immunoassay, Igaku Shoin).

Eiji Ishikawa et al., 1978) and horseradish peroxidase (manufactured by Sigma) using the periodic acid method (same document).
A solution of 20 times diluted conjugate (80 μ/ml) with buffer B (100 iIf!) was added to each well. After standing at room temperature for 2 hours, add buffer B 150
Washed three times with jt1. Next, 100 g of ortho-phenylenediamine substrate solution (1/15M sodium phosphate buffer containing 0.02% ortho-phenylenediamine and 35mM hydrogen peroxide, pH 7.2) was added to each well, and the mixture was allowed to stand at room temperature for 15 minutes. did. 2N H2SO*
50. d was added to each well to stop the reaction, and after leaving it at room temperature for 10 minutes, the absorbance (0, D, , and the antibody value was obtained from the difference.

All tests were conducted in two series, and the average value was taken as the measured value.

(2) Clinical test 29(1): 91-9 using Eisai's Atest ATL and the same sample as in Section 1.
4 (1985) (Eisai method), anti-ATLV antibody detection was performed on the same sample as in Section 1.

(3) The average values and standard deviation values of the measured values (0, D, ) measured in Sections 1 and 2 for the serum of 30 normal subjects were as follows.

gag-env O,055±0.013P-24
0,010±0.004 Eisai 0.040±0.009 If the cutoff value is the sum of the average value and twice the standard deviation value, P
-24 is 0.018.

0.081 for gag-env protein. In the case of Eisai, the cutoff value is 0.059.

Table 1 shows the measurement results for 57 ATL patients or HTLV-1 carriers.

Table 1 Table 2 shows the results of calculating the positive rate using the measured values in Table 1 and the above cutoff values.

Table 2 According to Table 2, for normal people, there were 0% false positives for all methods, and for ATL patients or HTLV-1 carriers, 10 people had false negatives for P-24 (cutoff value 0.01).
8 or less).

In the case of Eisai method, one person had a false negative result (cutoff value 0.05)
In contrast, in the case of gag-env protein, there was no false negative (cutoff value of 0.081 or less), and 10
It can be seen that the positive rate is 0%.

The results of calculations regarding the correlation between the Eisai method and the P-24 method or the gag-env method are shown in FIGS. 5 and 6. The correlation coefficient between Eisai method and P-24 method is 0.8294
．． The correlation coefficient between Eisai method and gag-any method is 0.74
It was 79. The value of this correlation coefficient is based on the Eisai method.

This shows that the P-24 method and the gag-env method have a mutual correlation.

Reference Example 1゜(1) Isolation and Purification of Plasmid pKYP26 and pEFM2 Escherichia coli [:Bscherichia] containing pKYP26
coli IKYP26 (FERM BP-86
3)] and pEFM2-containing Escherichia coli (Escher
ich 1acoli EEFM2 (ATCC53
228)) in 10+ mL of L medium (1% Batato tryptone, 0.5% yeast extract, 1% NaCCl (7,5) containing 50 μ/ml of ampicillin at 37°C for 18
Cultured for hours. The entire amount of this culture solution was inoculated into medium 11 containing 50 μ/ml of ampicillin, and cultured at 37°C.

After 4 hours, add chloramphenicol to 170μ/ml.
,! : and further cultured at 37°C for 16 hours. Bacteria were collected by centrifugation (5, OOO rpm for 10 minutes), and after washing the bacterial bodies with 0.8% NaCl,
QmM) Lith-hydrochloric acid (pH 8,0) was suspended in 20 ml and cooled on ice. Add 3 ml of 10 mg/ml Norizozyme and let it stand in water for 10 minutes, then add 0.5 M EDTA to 9. (
iml, let stand in water for 10 minutes, and add 2% Triton
100 (manufactured by Wako Pure Chemical Industries, Ltd.) was added thereto, and the mixture was left standing in water for an additional hour. 50,000 x g at 4°C;
Ultracentrifugation was performed for 1 hour, and about 4 Qml of supernatant was obtained.

Next, 3M NaOH was added to this supernatant to adjust the pH to 12.5.
The mixture was stirred gently at room temperature for 10 minutes. 2M) lithic hydrochloric acid (pH 7.5) was added to return the pH to 8.5, and the mixture was further stirred for 3 minutes.

At this point, the volume of the liquid was approximately 55 ml.

1/9 volume of 5M NaCj! after adding 10mM)
Lith-hydrochloric acid (pH 7.5) and phenol saturated with 1mM EDTA were prepared in advance, stirred vigorously, and then subjected to low-speed centrifugation (3.30 rpm).

The aqueous layer was collected under the same conditions for 10 minutes (hereinafter this process will be abbreviated as phenol extraction). 1/, 250 volumes of 5 mg/ml RNase A (manufactured by Sigma) was added, and an RNA degradation reaction was performed at 37°C for 1 hour.
5 volumes of 5M NaCj! Add 1/3 volume of 30%P
Add EG6000 (manufactured by Hanui Chemical Co., Ltd.) and heat to 20°C.
Let it stand for a while. The precipitate was collected by centrifugation, dissolved in 2 ml of a solution consisting of 10 mM) Lis-HCl (pH 7.5) and 1 mM EDTA, added with sodium dodecyl sulfate (SDS) to a concentration of 0.5%, and mixed with Protei.
Nase K (manufactured by Sigma) was added at a concentration of 50 μ/ml, and a proteolytic reaction was carried out at 37° C. for 1 hour.

After repeating the phenol extraction three times, an equal amount of chloroform was added, and after vigorous stirring, the aqueous layer was collected by low-speed centrifugation. 1/10 volume of 3M sodium acetate was added, 2.5 volumes of ethanol was added, and the mixture was left standing at -20°C for 1 hour.

Refrigerated centrifugation method (4°C, 11,000 rpm,
10 min) to collect the precipitate and 10mM)! JS hydrochloric acid (pH
7,5) and 1 ml of a solution consisting of 1 mM EDTA. In this way pKYP26 and pEFM
We were able to get 800 shoulders each for 2. The structure of pKYP26 is EcoRI.

Kpn 1. BamHI, Bgj7I[, Ps t 1
and confirmed by agarose gel electrophoresis. Also p
The structure of EFM2 is Xho I.

KpTl 1. BgI! Il, Hpa 1. Ps
It was cleaved with tI and confirmed by agarose gel electrophoresis.

(2) Plasmid DNA containing ATLV P24 gene
Construction (2)-L Construction of pAFB7: pGELI Cε5cherichia coli I
[Collected from GFiLl (FERM BP-629) by a conventional method] 1QmM) Lith-hydrochloric acid (pH 7.5),
7mM MgCl1. ．． A solution containing 6mM mercaptoethanol and 100mM NaCl (hereinafter referred to as Y-1
0011 solution) Dissolve in 40ρ, add restriction enzyme P
10 units of st■ (manufactured by Takara Shuzo Co., Ltd.; unless otherwise specified, restriction enzymes manufactured by Takara Shuzo Co., Ltd. were used) and 3 units of BamHI were added, and a digestion reaction was carried out at 37°C for 2 hours. From this reaction solution, L
Approximately 1.700 including βpp terminator by GT method
Approximately 0.2 bp DNA fragment was obtained.

Separately, add 10■ of pGEL 1 to 100, Y of d
-100 buffer, added 16 units of restriction enzyme PstI, and incubated at 37°C.

Digestion reaction was carried out for 1 hour. After confirming complete Pstl digestion by agarose gel electrophoresis, 3 units of restriction enzyme Hpa 1 was added and the digestion reaction was carried out at 37°C for 30 minutes to partially degrade Hpa 1. From this reaction solution, approximately 94 cells containing the trp promoter were extracted using the LGT method.
Approximately 0.2 Jtg of a 0 bp partially degraded DNA fragment was obtained.

Next, 30 g of pAFAlo (the plasmid shown in Figure 1 carried by ATCC39582) was added to 2504 Y-
100 buffer, add 140 units of restriction enzymes Hpa I and 30 units of BamHI, perform a digestion reaction at 37°C for 3 hours, and use the LGT method to extract a DNA fragment of about 700 bp containing the P24 gene from the reaction solution. 5 ■I got it.

About 0.1 g of the PstI-BamHI fragment (about 1,700 bp) derived from pGELl obtained above, PstI-)
(par fragment (about 940 bp) about 0.15xr and HpaI-BamHE fragment from pAFA 10 (about 7
00 bp) approximately 0.2■ to 20 mM) lithium-hydrochloric acid (
pH 7.5).

A solution containing 10mM MgCA'2.10mM dithiothreitol and 1mM ΔTP (hereinafter referred to as T4
DNA! J gauze buffer) was dissolved in 20ρ, and 3 units of T4DNA ligase (manufactured by Takara Shuzo Co., Ltd.) was added.
The binding reaction was carried out at 4°C for 16 hours.

The reaction mixture was used to transform Escherichia coli strain 88101 [Polliver et al., Gene 2, 75 (1977)] to obtain colonies of A p 1, which were then transformed using the method of Birnboim et al. [Nuclectric Acid Research, 1513 (1979)]. )] to recover plasmid DNA, and pAFB7 shown in FIG. 1 was obtained.

(2) Construction of -2pAAB6: pTAG424A (plasmid shown in FIG. 2 carried on FERM BP-341, JP-A-660-6153)
4) 21L was dissolved in 20ρ Y-100 buffer, 4 units of restriction enzyme BamH1 was added, and a digestion reaction was performed at 37°C for 2 hours.

Subsequently, the NaCl concentration of the solution was adjusted to 150 mM, and 4 units of the restriction enzyme Nco I (manufactured by Oppon Gene Co., Ltd.) was added to perform a digestion reaction at 37°C for 2 hours. Next, BamH
The DNA cut with I, Nco (33mM) lis-acetic acid (pH 7,9>).

66mM potassium acetate, 10mM magnesium acetate, 5
mM dithiothreitol, and 0.4 mM dA
TP, dCTP, dGTP.

It was dissolved in 20 g of a solution containing dTTP (hereinafter abbreviated as T4 DNA polymerase buffer), 1 unit of T4 DNA Δ polymerase (manufactured by Takara Shuzo Co., Ltd.) was added, and a reaction was carried out at 37° C. for 30 minutes.

Dissolve the above enzyme treatment solution in 204 T4 DNA ligase buffer, add 3 units of T4 DNA ligase, and heat at 4°C.
The binding reaction was carried out for 18 hours.

E. coli strain 88101 was transformed using the reaction solution, and A
A colony of pl was obtained, and plasmid DNA was recovered from this colony by the method of Birnboim et al. to obtain pAAB6 shown in FIG.

(2) Creation of -3pAHAl: 15Jtg of pAAB6 obtained in the previous section was added to 1004 Y-1
Dissolved in 00 buffer, 5tu115 units of restriction enzyme and Cj
! aI (2, - England Bio-Lapse (N
EW Bngland Bio Labs) 20 units were added thereto, and the mixture was heated at 37°C.

Digestion reaction was carried out for 3 hours, and p2 was extracted from this reaction solution by LGT method.
About 0.3 μ of a DNA fragment of about 400 bp containing the first half of 4 genes was obtained.

Next, 5■ of pGEL1 was dissolved in 404 Y-100 buffer, and the restriction enzyme Cj! New England Bio Labs
) and 8 units of PSt were added, and a digestion reaction was carried out at 37°C for 2 hours. From this reaction solution, trp is extracted using the LGT method.
Approximately 1.000 bp of D containing part of the promoter of the system
Approximately 0.2 inch of NA fragment was obtained.

Separately, the two shoulders of pKYP26 obtained in Section 1 were lo
mM) Squirrel-HCj! (pH 7,5).

A solution containing 7mM MgCRz, 6mM+1 captoethanol and 10mM NaCl (hereinafter referred to as Y
-10 buffer) Dissolve in 30ml of restriction enzyme K.
Five units of pnI were added and the digestion reaction was carried out at 37°C for 2 hours.

The DNA cut with Kpn I was converted into 204 T4 DNA.
T4 DNA polymerase 1 dissolved in polymerase buffer
Add units and 37°C.

The reaction was carried out for 30 minutes. This DNA reaction solution was dissolved in 30/1100 buffer solution, 14 units of restriction enzyme pst was added, and the mixture was incubated at 37°C.

Digestion reaction was carried out for 2 hours. From this reaction solution, z was obtained using the LGT method.
Approximately 1.700 b including a portion of the PP terminator
Approximately 0.2 μ of DNA fragment of p was obtained.

About 1 g of the 5tul-C1la fragment (about 400 bp) derived from pAAB6 obtained above.

Cj from pGEL1! aI-PstI fragment (approximately 1,0
00bp) approximately 0.2 shoulder, KpnI- from pKYP26
PstI fragment (about 1.700 bp) about 0.1 d
of T4D in 4 units of T4D ligase buffer.
NA ligase was added and the binding reaction was carried out at 4°C for 18 hours.

E. coli strain 88101 was transformed using the reaction solution, and Δ
A colony of p1 was obtained, and plasmid DNA was recovered from this colony by the method of Birnboim et al. to obtain pAHAl shown in FIG. 2.

(2) Creation of FGIO to -4p: 511g of pAHAl obtained in the previous section was added to 50d of Y-1,0
0 buffer, add 8 units of restriction enzyme PStI, and add 3
Digestion reaction was carried out at 7°C for 2 hours. From this reaction solution, LGT
By this method, about 0.5 jtg of a DNA fragment of about 1.100 bp containing a part of the TRP promoter and the N-terminal part of the P24 gene was obtained.

Next, 6 ml of pAFB7 constructed in Section 1 was dissolved in 30 d of y-too buffer, 8 units of restriction enzyme BamHI was added, and the mixture was incubated at 37°C.

Digestion reaction was carried out for 2 hours. 0.2M trif, -HCji! to this reaction solution. (+)88.0), 120mM
Ca Cj! 2, 120 m M M g C
A solution consisting of β2,2M NaC1 and 10mM EDTA! In! Add 154ml of sterile water, and heat to 30°C for 30 minutes.
It was kept warm for minutes. BAL31 nuclease [Bethesda Research Laboratories (Bethesda Re5e)
Arch Laboratories, Inc.] was added thereto, and a decomposition reaction was carried out at 30° C. for 80 seconds. After the reaction, phenol extraction was performed, and DNA was recovered by ethanol precipitation.

This DNA was dissolved in 3040Y-100 buffer, 8 units of restriction enzyme PstT was added, and a digestion reaction was performed at 37°C for 2 hours. From this reaction solution, about 0.1 inch of a DNA fragment of about 450 bp containing the P2424 gene was obtained by the LGT method.

PstI fragment derived from pAHA1 obtained above (approximately 1.10
0bp) approximately 0.2■, derived from pAFB7 (7) Ps t
I-BAL3 L nuclease digested fragment (approximately 450 bp
)0.1■, Kp derived from pKYP26 obtained in the previous section
n r-PstI fragment (approximately 1.700 bp) 0
．． Dissolve one fish in 304 T4 DNA ligase buffer,
Six units of T4 DNA ligase were added, and the binding reaction was carried out at 4°C for 18 hours.

E. coli strain 88101 was transformed using the reaction solution, and A
A colony of pl was obtained, and plasmid DNA was recovered from this colony by the method of Birnboim et al. to obtain pAFG 10 shown in FIG. The nucleotide sequence of the C-terminus of the P24-encoding region was determined, and as shown in Figure 3, it includes up to the C-terminus of P24, and also contains
It was confirmed that the structure had 2.Leu-3er.Δsn.

(3) Construction of a recombinant pair plasmid pETI7 encoding a hybrid antigen polypeptide in which the antigen polypeptide encoded by the gag gene of ATLV and the antigen polypeptide encoded by the env gene are fused: The assembly obtained in Section 2 10 of recombinant plasmid pAFG10
was dissolved in 1004 Y-100 buffer and added with restriction enzyme Xh.
10 units of oI.

12 units of 5tuI were added and the digestion reaction was carried out at 37°C for 3 hours.

From this reaction solution, approximately 630 bp of D containing the tributophan promoter and the first half of the P24 gene was extracted using the LGT method.
Approximately 0.5 inch of NA fragment was obtained.

Next, 5μ of pEFM2 obtained in Section 1 was added to 50 Y-10
0 buffer, 10 units of restriction enzyme XhoI, Hp
8 units of aI were added and the digestion reaction was carried out at 37°C for 3 hours.

From this reaction solution, about 1 inch of DNA fragment of about 2.700 bp containing the latter half of the env gene was obtained by the LGT method.

0.05 pmole of the XhoI-3tuI fragment (approximately 630 bp) derived from pAFG10 obtained above and the Xho I-Hpa I fragment (approximately 2,700 bp) derived from pEFM1.
．． Dissolve 01 pmole in 40ρ T4 DNA ligase buffer, add 5 units of T4 DNA ligase, and incubate at 4°C for 1 hour.
The binding reaction was carried out for 8 hours.

E. coli strain 88101 was transformed using the reaction solution.A
A colony of pl was obtained, and plasmid DNA was recovered from this colony by the method of Birnboim et al. to obtain pETr7 shown in FIG.

(4) Production of a fusion polypeptide between the antigen polypeptide encoded by the gag gene and the antigen polypeptide encoded by the env gene by E. coli harboring pETI7: Recombinant plasmid pE obtained in Section 3.3
Escherichia coli W3110StrA strain (
FERM BP-732) was transformed. The obtained Ap3 colonies were placed in 8 ml of MCG medium [0.6% H
a 2 HP 04.

0.3% KH2PO4,0.5% NaCj7゜0.
It was inoculated into 1% NH, cf, 0.5% glucose, 0.5% casamino acid, 1mM MgS○4+ 4μg/ml vitamin Bl+ pH 7,21, and cultured at 30°C for 18 hours. The obtained culture solution was heated at 8.000 rpm, 1
The cells were collected by centrifugation for 0 minutes. The cells were suspended in Laemmli's sample buffer, subjected to 5DS-polyacrylamide gel electrophoresis, and stained with Coomasieve IJ IJ Ant Blue, with a molecular weight of approximately 25. A polypeptide band was detected at the OOO site. This band was not present when E. coli not containing the plasmid was used.

The molecular weight of this polypeptide was consistent with the molecular weight of the hybrid antigen polypeptide (25,051,02) predicted from the structure of plasmid pETI7.

(5) Centrifugation (8,0
0 Orpm, 30 minutes), and the resulting bacterial cells were incubated at 20 Q
ml of deionized water and a MANTON-GAULIN homogenizer (manufacturer; MANTON-GAULIN M).
AN(IPACTURING Co., lNC
6USA) Bacterial cells were disrupted using a pressure of 400 kg/cd, and a precipitate fraction was obtained by centrifugation. This precipitated fraction was analyzed by 5DS-polyacrylamide electrophoresis and was found to contain 845 gag-env proteins.

This precipitate fraction was mixed with 4% (W/V) Lydon X-100,
20 containing 10mM sodium ethylenediaminetetraacetate
mm! A precipitate fraction was obtained again by dispersion in J acid buffer (pH 7,4) and centrifugation, and this precipitate was mixed with 2mM sodium ethylenediaminetetraacetate, 0.3% (V/V) 2-mercaptoethanol, and 7M urea. Phosphate buffer (pH
7,4) and 20mM containing 7M urea in advance.
! J acid buffer (hereinafter referred to as equilibration buffer, pH 7,
TSKge ICM) Yopar 650 equilibrated with 0)
(manufactured by Toyo Soda Kogyo Co., Ltd.) 10 Qml was passed through the tower and adsorbed.

After washing the column with equilibration buffer, 0. Elution was performed with equilibration buffer to which IMNaCl was added to obtain a high purity fraction of 25 Qml. This fraction contained gag-env protein of 430 axes. This high purity fraction was added to 1QmM carbonate buffer (pH
9,4) Dialyzing 31 as an external solution1. After removing the resulting precipitate by centrifugation, the centrifugation supernatant was used as the target gag-e.
40 Qml of nv protein solution was obtained.

S D S - P A G E 1. Further analysis showed that the protein purity was 90% or more, and the yield was 160 mg.

Effects of the Invention According to the present invention, anti-ATLV antibodies can be detected with high accuracy, and serodiagnosis of ATL can be performed effectively. Furthermore, the method of the present invention can be applied to diseases that exhibit cross-reactivity with P-24.

[Brief explanation of the drawing]

FIG. 1 shows the construction process of plasmid pAFB7. FIG. 2 shows the construction process of plasmid pAHA1. FIG. 3 shows the construction process of plasmid pAFG10. FIG. 4 shows the construction process of plasmid pETI7. FIG. 5 shows a correlation diagram where the Eisai method antibody values are taken as the Y axis and the P-24 method antibody values are taken as the X axis. The correlation coefficient is 0.
It is 8924. Using the least squares method (linear regression) using a single frequency distribution, the regression equation is determined as Y=2.7437X+
'0.2151 is shown. Figure 6 shows the Eisai method antibody values on the Y axis, gag-env
A correlation diagram is shown when the X-axis is the antibody value of the method. The correlation coefficient is 0.7479. When calculating the regression equation using the least squares method (linear regression) using a single frequency distribution, Y = 1.9473
Indicates X-0,1119.

Claims (4)

[Claims] (1) Immunological method using as an antigen substance a polypeptide encoded by a fusion gene in which the gag gene of adult T-cell leukemia virus (hereinafter referred to as ATLV) or a part thereof is combined with the env gene of ATLV or a part thereof 1. A method for detecting anti-ATLV antibodies, which comprises detecting anti-ATLV antibodies in a sample. (2) Immunological methods include radio immunoassay, enzyme immunoassay, fluorescence immunoassay, reverse passive agglutination, phytohematoaggregation, particle agglutination, laser nephrometry, and immunohemolysis (using red blood cells or liposomes). Detection method according to claim 1, which is selected from the following methods: (3) The detection method according to claim 1, which uses an antigen substance coated on a solid phase. (4) The detection method according to claim 3, wherein the solid phase is a synthetic resin, a glass tube, glass beads, or a glass microtiter plate.